Abstract

OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte
plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has
not been reported but may be revealed under conditions of insulin resistance.

RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western
blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were
respectively estimated by oral glucose tolerance test and secretion of [U-14C]-3-O-methyl glucose into lumen.

RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects
but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular
accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in
39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited
endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen.

The intestinal tract is determinant in energy homeostasis through control of sugar absorption and gut hormone release during
digestion (1–4). Accordingly, the regulation of nutrient absorption has implications in metabolic diseases and their increasing prevalence
worldwide.

Sugar absorption relies on the coordinated functions of transporters at the surface membrane of enterocytes. In the apical
plasma membrane, the high-affinity Na-coupled cotransporter SGLT1 performs glucose and galactose extraction from the lumen
(2) and GLUT5 transports dietary fructose (5). In the basolateral membrane, GLUT2 provides an exit pathway (6,7). These transporters are expressed in the duodenum and jejunum and at lower levels in the ileum. In rodent intestine, GLUT7,
a high affinity transporter for glucose and fructose, was identified in the apical membranes of ileal enterocytes and colonocytes
(8). Rodent models have shown that GLUT2 can be inserted into enterocyte apical membranes in response to oral glucose or fructose
(9,10). This result constitutes an adaptation process to complement SGLT1 and GLUT5 uptake capacities when dietary sugar intake
is high (10). Apical GLUT2 translocation is linked to dietary sugar concentration in the lumen and is reduced by fasting (10,11). Apical GLUT2 has been identified in adult and neonate rodent enterocytes as well as in insects, sheep, and pigs (rev. in
12,13). Although different signaling mechanisms have been reported to promote insertion of GLUT2 into apical membranes of enterocytes
(rev. in 12), only insulin has been shown to trigger GLUT2 internalization, thereby slowing sugar uptake in the intestine during digestion
(14). The relevance of this mechanism in the human small intestine deserves investigation. However, GLUT2 trafficking in human
enterocytes is supported by studies in enterocytic Caco-2/TC7 cells (14,15). Ethical considerations render it difficult to directly study the impact of sugar on enterocyte GLUT2 location in humans.

In mice, insulin resistance maintains GLUT2 in enterocyte apical membranes, thereby creating conditions for increased dietary
sugar uptake (14). Furthermore, experimental diabetes in rats with insulinopenia and hyperglycemia provokes mucosal hypertrophy and increases
mRNA and protein expression of GLUT2, GLUT5, and SGLT1 (16). In humans, obesity is characterized by the development of insulin resistance and type 2 diabetes (17–20). However, apical GLUT2 was not found in duodenal biopsies of overweight human type 2 diabetic subjects (21).

Insulin sensitizers are used in the treatment of type 2 diabetic subjects. In rodents, metformin increases intestinal sugar
use (22,23) and expression of SGLT1 and GLUT5 (24) and it decreases glucose absorption (25). Metformin also promotes apical GLUT2 location in rodent enterocytes via AMP-activated protein kinase (AMPK) (26). In the human intestine, the effects of metformin on GLUT2 location have not yet been reported.

Bariatric surgery is a therapeutic option to reduce obesity with a curative potential for severe metabolic disorders (27). In jejunal samples obtained during bypass surgery of morbidly obese subjects, changes in GLUT2 location in enterocytes
are expected according to the metabolic status of subjects. In the current study, morbidly obese subjects were carefully characterized
for history of obesity, comorbidities, treatments, and dietary composition from questionnaires. GLUT2 location in jejunal
enterocytes of obese and lean control subjects was assayed, and links with bioclinical parameters were analyzed. The consequences
of insulin resistance, diabetes and dietary habits on intestinal function were revealed from comparison with lean subjects.
The impact of metformin treatment and high-fat diet on GLUT2 distribution were explored in genetically obese and wild-type
mice, respectively.

RESEARCH DESIGN AND METHODS

Human obese and lean subjects.

Morbidly obese subjects (n = 62) involved in a gastric surgery program were recruited (2006–2008) in the Nutrition Department, CRMO, Hôtel-Dieu Hospital,
Paris, France. Subjects (n = 14 for men; n = 48 for women) were aged between 19 and 64 years and met criteria for bariatric surgery: BMI ≥40 kg/m2 (90%) or BMI ≥35 kg/m2 combined with at least one comorbidity (type 2 diabetes, hypertension, obstructive apnea syndrome, or dyslipidemia). On the
basis of the dietary food questionnaires, subjects could be defined as high-fat eaters when ingesting >30% of calories as
lipid and low-carbohydrate eaters when ingesting <50% of calories as carbohydrate. Lean subjects (n = 7) were selected from a set of age-paired (range 17–68 years) normal-weight nondiabetic individuals. Subjects were fasted
as required for the surgery.

Jejunal samples of obese and lean subjects.

A tissue bank was made with jejunal samples usually discarded during Roux-en-Y gastric bypass. Samples were fixed in alcohol-formalin-acetic
acid (AFA) after excision and imbedded in paraffin wax by pathologists. Some samples were snap-frozen in liquid nitrogen for
biochemical investigations. Jejunal biopsies taken during routine gut exploration of seven lean subjects were obtained by
double-balloon explorative enteroscopy and sent to the pathology department.

Ethics statement.

The Ethics Committee of Hôtel-Dieu Hospital approved the clinical investigations for both obese and lean subjects who gave
written informed consent. Experiments with mice received approbation by the local ethics committee of Université Pierre et
Marie Curie (UPMC) for animal use (p3/2008/042).

Statistical analysis.

Results are expressed as mean ± SEM, and the significance between the mean of continuous parameters was evaluated by the Mann-Whitney
statistical test using the StatEL or R statistics software packages (http://www.r-project.org). Contingency tables were performed for analysis of categorical data. The χ2 statistics tests (or Fisher exact test for small sample size) were used to test the independence of GLUT2 localization and
the other categorical data. All probabilities were two-tailed with significance set at P < 0.05.

Most diabetic obese subjects took metformin (74%) or insulin (48%) in various combinations with other oral antidiabetic drugs
(OADs). Although observed in a few subjects, 65% of metformin-treated diabetic obese subjects and all obese subjects taking
insulin and metformin exhibited apical GLUT2 (Supplementary Table 3). However, the effects of metformin alone or in combination could not be established unequivocally, since only 18% of diabetic
obese subjects received metformin as single antidiabetic therapy. Ob/ob mice were therefore used to assess the effects of metformin on GLUT2 location.

Endosomal accumulation of GLUT2 in enterocytes of morbidly obese subjects.

The number of obese subjects exhibiting endosomal GLUT2 accumulation revealed a link with the macronutrient composition of
their diet (Fig. 2C) but not with total calorie intake (Supplementary Table 2). Indeed, 83% of subjects exhibiting endosomal GLUT2 accumulation were high-fat eaters (black columns; P = 0.04) versus 55% of subjects with other GLUT2 locations. Endosomal GLUT2 accumulation was also related to lower carbohydrate
consumption (white columns; P = 0.04). We next used a mouse model to test the hypothesis that unbalanced carbohydrate/high-fat diets could promote GLUT2
accumulation above the nucleus.

Impact of diet and metformin on GLUT2 location in the mouse enterocyte

Endosomal GLUT2 accumulation in enterocytes of HFLC-fed mice.

Mice fed an HFLC diet for 12 months were insulin resistant but had similar weight gain as control mice (M25 diet) (Table 2). OGTTs revealed the progress of glucose intolerance (Fig. 3A) and significant increase of fasting blood glucose concentration (Fig. 3B) with time of exposure to HFLC diet when compared with controls. As anticipated, GLUT2 was found in basolateral membranes
in healthy control mice (Fig. 3C). In contrast, all HFLC mice exhibited endosomal accumulation and significantly lower levels of basolateral GLUT2 in enterocytes
(Fig. 3D). These GLUT2 locations resemble those of obese subjects eating unbalanced fat/carbohydrate diets.

Endosomal accumulation of GLUT2 in mice fed a high-fat diet. Mice were fed either the control chow diet (M25, white symbols) or HFLC diet (black symbols) for up to 12 months. A: OGTTs are shown after 2 months (diamonds) and 12 months (triangles) in two groups of eight mice fed the control or HFLC diet. B: Fasting blood glucose concentration increases were measured with time (mg/dL ± SEM; n = 8; ***P < 0.001). C and D: Representative confocal images of GLUT2 (green) and Na,KATPase (red) location in 6-μm sections of control and HFLC jejuna after 12 months. Scale 10 μm. Note the endosomal accumulation of GLUT2
(arrows) and low basolateral GLUT2 in the jejunum of HFLC-fed mice compared with control diet mice. E: Initial slopes (T15/T0) of blood glucose concentration after OGTT (**P < 0.01) estimating sugar absorption. In F, quantification of GLUT2 abundance by Western blot (density arbitrary units ± SEM, **P < 0.01) is shown in postnuclear membrane preparations of control and HFLC jejuna. (A high-quality digital representation
of this figure is available in the online issue.)

In HFLC mice, glucose absorption decreased slowly, as indicated by the significantly lower initial slope of blood glucose
concentration during OGTT (Fig. 3E), i.e., basal glucose concentration increased (Fig. 3B) with essentially a similar 15-min glucose concentration in OGTT. As expected from a low-carbohydrate dietary supply, the
protein abundance of GLUT2 in jejuna was reduced in HFLC-fed mice compared with control mice (Fig. 3F). Sequestration of GLUT2 in intracellular compartments and lower levels of GLUT2 expression could explain the lower rates
of glucose absorption.

In obese ob/ob mice and lean ob/+ littermates, the impact of metformin on GLUT2 locations in enterocytes was tested. The HOMA-IR value was 38-fold higher
in ob/ob mice than in ob/+ mice (Table 2), indicating a strong insulin resistance. In agreement with this finding, insulin concentration at 30 min during the OGTT
was ninefold lower in ob/+ mice (Table 2). As expected in ob/+ mice, GLUT2 was only in the basolateral membrane of enterocytes, whereas ob/ob mice exhibited permanent apical and basolateral GLUT2 (Fig. 4A).

DISCUSSION

Most morbidly obese subjects (76%) in this study exhibited apical GLUT2 in the fasting state, which contrasted with its basolateral-only
location in lean subjects. To the best of our knowledge, this is the first demonstration of an apical GLUT2 location in the
human small intestine. We also discovered that GLUT2 could accumulate in the endosomes of 39% obese subjects, thus revealing
the complexity of obesity-related adaptation in the intestine.

To understand the underlying mechanisms that alter GLUT2 trafficking in human enterocytes, associations were established between
GLUT2 location and the clinical parameters of well-phenotyped obese subjects, such as degree of insulin resistance and unbalanced
dietary intake. The correlation between fasting blood glucose concentration and percentage of subjects with apical GLUT2 indicated
that this location is linked to changes in glucose homeostasis or diet-induced insulin resistance in humans. Indeed, in hyperglycemic
(400 mg/dL) hypoinsulinemic diabetic rats (32) as well as in hyperglycemic (110 mg/dL) hyperinsulinemic mice (14), GLUT2 was observed in the apical membrane of jejunal enterocytes, indicating complex interplay between these parameters
to control GLUT2 location. Apical GLUT2 has not been observed in duodenal biopsies from diabetic subjects (21) in contrast to the 73% of obese diabetic subjects displaying apical GLUT2 in the current study. The divergence in results
may be related to subject obesity, the intestinal segment studied (duodenum vs. jejunum), or antidiabetic treatments.

The second major observation in 39% of the human obese subjects was the accumulation of GLUT2 in compartments containing the
EEA1 endosomal marker. There was a significant association between EEA1/GLUT2 accumulation and HFLC diets. In mice fed an
HFLC diet for 5 months (resulting in strong insulin resistance without obesity [14]), we have previously reported apical GLUT2 accumulation in jejunal enterocytes. Prolonged feeding with HFLC for up to 12
months promoted GLUT2 accumulation above the enterocyte nucleus, suggesting that trafficking defaults were further aggravated.
Thus, long-term exposure to unbalanced high-fat diets may similarly affect human intestinal function. High-fat diets were
reported to alter GLUT2 trafficking in mouse pancreatic β-cells and to promote intracellular GLUT2 location (33). In mouse pancreatic β-cells, high dietary fat was shown to reduce GLUT2 glycosylation from lower GlcNacT-IV transferase
(MGAT4a) expression and impaired GLUT2 trafficking into plasma membranes (34). In the current study, MGAT4a protein was reduced twofold in obese subjects exhibiting endosomal versus apical GLUT2. This
finding suggests that intestine and pancreatic β-cells share similar mechanisms for glycosylation-related GLUT2 trafficking.
In insulin-secreting Min6 cells, high glucose concentration is suspected to trigger GLUT2 degradation in lysosomes (35). In contrast, we did not observe GLUT2 accumulation in lysosomes (LAMP-1) in any of the obese subjects, suggesting that
GLUT2 degradation was unaffected. Our findings therefore indicate that macronutrient balance in diet is a controlling factor
of GLUT2 trafficking into enterocytes.

Metformin, an antidiabetic biguanide drug in current clinical use, promotes a dramatic increase of glucose utilization in
human jejunum (36) and in rat intestine (23,37). In rodents, the drug accumulates in the mucosa (38), where it increases SGLT1 and GLUT5 expression, leaving GLUT2 mRNA levels unaltered (24). The effects of metformin on glucose homeostasis have been related to AMPK, increasing glucose utilization, and reducing
hepatic glucose output (39). AMPK with 5′-aminoimidazole-4-carboxymide-1-β-d-ribofuranoside (AICAR) recruits GLUT2 in enterocyte apical membranes in rodents (26). This result raises the issue of increasing apical GLUT2 location using pharmacological agents as a means to gain control
on intestinal absorption and blood glucose levels. In this study, the 65% of subjects on metformin exhibited apical GLUT2,
suggesting that the drug may also modify intestinal sugar transepithelial fluxes. However, a study to address the multiple
associations of antidiabetic drugs on intestinal GLUT2 location requires a large number of diabetic subjects.

The harm or benefits of apical GLUT2 location is schematized in Fig. 5. Under fasting conditions, SGLT1 captures any trace glucose from the lumen. GLUT2 in the basolateral membrane provides a
glucose entry pathway to fulfill enterocyte metabolic needs. We showed that the transient insertion of apical GLUT2 in healthy
enterocytes, occurring after a sugar-rich meal, triples the initial rate of sugar uptake (10). The magnitude of postprandial hyperglycemia caused by rapid entry of sugar into the bloodstream will modulate pancreatic
insulin secretion. The action of insulin will then contribute to internalize apical GLUT2 and promote a return to basal location
and function (14). We showed in mouse that pathological and permanent apical GLUT2 in obesity favors glucose release from the mucosa into
the lumen. Glucose efflux mediated by permanent apical GLUT2 will occur as soon as the glucose gradient between blood and
lumen is reversed. This efflux into the lumen will maintain an abnormal glucose provision to fuel bacteria metabolism. This
result may contribute to changes in gut microbiota, as observed in obese mice (40). We anticipate that permanent apical GLUT2 accelerates glucose uptake immediately after a sugar-rich meal in obese subjects
as well as in lean subjects. The consequences of permanent apical GLUT2 on the net intestinal sugar absorption need to be
quantified in more detail.

GLUT2 trafficking in cell types other than enterocyte and kidney cells is not fully understood. In the rat liver, GLUT2 and
insulin receptors are internalized as a complex that was proposed as a means to accelerate insulin inhibition of hepatic glucose
production (41). In rodent pancreatic β-cells, GLUT2 location depends on the abundance of glucose in the diet (34) or in the culture medium of cell lines (35) and could modulate insulin secretion. In the mouse brain, GLUT2 is involved in the regulation of food intake (42,43). A coordinated regulation of GLUT2 trafficking in these tissues might implement a gut-brain axis control on glucose homeostasis.

Our study reveals that GLUT2 can be inserted into the apical membrane of human enterocytes. In morbidly obese subjects, GLUT2
accumulated in apical and endosomal membranes. These pathological locations have been respectively linked to metabolic alteration
and nutritional patterns. Transepithelial glucose exchange is favored by apical GLUT2 and reduced by endosomal GLUT2. Apical
GLUT2 could be triggered by drugs such as metformin to provide a glucose exit pathway into the lumen contributing to the drug
hypoglycemic effect. Apical GLUT2 can therefore provide plasticity to sugar absorption and constitute a compensatory/adaptive
mechanism to limit the magnitude of hyperglycemia. Altered GLUT2 locations in enterocyte are a sign of intestinal adaptation
to human metabolic pathology.

ACKNOWLEDGMENTS

The work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Pierre and Marie Curie (UMPC)
P6 and René Descartes P5 universities, and the Centre National de la Recherche Scientifique (CNRS). A.C. and J.G. hold postdoctoral
fellowships from Agence Nationale de la Recherche-Alimentation et Industries Alimentaires (ANR-ALIA 007-01).

No potential conflicts of interest relevant to this article were reported.

A.A.-O., M.M.-S., and K.G. performed the immunolocalizations. C.P., A.C., and J.T. recruited subjects and performed the phenotype
and statistical analysis. N.V. and D.H. did tissue sampling and processing. D.C. was in charge of electron microscopy and
supervised K. Bourhaba. M.L.G., A.H., and P.S. contributed to biochemical studies in mouse and human intestines. J.G., A.La.,
and C.M. performed in vivo glucose efflux measures. K.C. supervised clinical studies and statistical analysis and contributed
to the writing of the manuscript. A.Le. and E.B.-L. initiated the study, designed experiments, supervised the analysis, and
wrote the manuscript.

Part of this work was presented at the Physiological Society Themed Meeting, Newcastle upon Tyne, U.K., 6–8 September 2009
and Epithelium and Membrane Transport Group, Salerno, Italy, 7–10 April 2010.